Recent changes in the security situation facing citizens in the United States and military personnel abroad have greatly increased the threat that chemical weapons will be used against American forces and American civilians in the field as well as at home. Current information indicates that bioterrorists have contemplated using organophosphate (OP) nerve agents. OP nerve gases are a threat to military personnel and civilians (e.g., Gulf War exposure to the military and Tokyo subway system exposure to civilians). In addition, farmers, agricultural workers and pesticide applicators handle large amounts of OPs and are potentially exposed to these toxic materials. Between 150,000 and 300,000 OP-related toxic incidences are reported annually in the United States (Rosenstock, Keifer et al. 1991). This situation points to an urgent need for an efficient, fieldable and inexpensive way to detect OP nerve agents. Available treatment of acute OP nerve agent poisoning only acts in a competitive fashion and is not adequate since it does not prevent neuronal brain damage and incapacitation. Detection instrumentation is an essential component of any protection paradigm, and thus a challenge is to develop decontamination and detection methodology for various OP agents (Chen and Mulchandani 1998; Sogorb and Vilanova 2002).
OP nerve agents act by inhibiting the cholinesterase (ChE) family of enzymes, mostly in the brain, central nervous system and blood. Within the cholinesterase family, acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) are the best known targets. In the case of AchE, the resulting OP adduct inactivates the enzyme, allows acetylcholine (Ach) to build up in the synapse, stimulate autonomic receptors, and block neuromuscular junction receptors. The symptoms resulting from nerve agent exposure are primarily the consequence of accumulation of excess Ach at nerve junctions where ordinarily small amounts of Ach are needed for impulse transmission. Non-cholinergic symptoms have been linked to OP exposure including delayed neuropathy, leukemia, depression, genotoxicity, pulmonary toxicity and vision loss. Evidence for non-AchE targets have been shown with AchE knock-out mice (Xie, Stribley et al. 2000; Duysen, Li et al. 2001).
Other proteins also form adducts with OPs. These proteins include, but are not limited to, serum albumin, transferrin, tubulin, carboxylesterase, acylpeptide hydrolase, fatty acid amide hydrolase, the cannabinoid CB 1 receptor, fatty acid synthase, dipeptidyl peptidase 9, prolyl oligopeptidase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase, (Tuin et al. Chem Res Toxicol. 2009). The biological effects of adduct formation with these other proteins are not fully understood.
A prominent enzyme for the peripheral hydrolysis of esters (and OP esters) in humans is butyrylcholinesterase (BuChE) also known as serum cholinesterase. BuChE is a glycoprotein of 4 identical subunits (Lockridge et al., 1987). Like the 3D structure of AchE from Torpedo californica, the active site of BuChE contains a traditional catalytic triad Ser198-Glu325-His338, and the active site of BuChE is believed to lie near the bottom of a deep and narrow gorge. The enzyme is of toxicological and pharmacological importance and thought to have a role in protection against poisons that are eaten or inhaled (Jbilo et al., 1994; Neville et al., 1990). BuChE scavenges low doses of OP and carbamate pesticides by forming covalent bonds with these agents through the active site serine and therefore protects humans from the toxic effect of these poisons (Lockridge and Masson, 2000). The initial interaction and multi-step subsequent reactions between OPs and ChE is illustrated in Scheme 1 (Masson, Fortier et al. 1997).
When an OP nerve agent reacts with AchE or BuChE, several OP-adducts are possible and the rate of covalent modification (or dealkylation) versus recovery, or aging versus reactivation, plays an important role in the potency and duration of toxicity (in the case of AchE). Most reactive OPs contain a dialkoxy phosphate or phosphonate, and a good leaving group X. The leaving group is generally a halogen, mercaptan (thiolester), phenoxy derivative, or other. The alkoxy phosphonates sarin and soman react with cholinesterase to afford phosphono-cholinesterase adducts after loss of F. Likewise, VX forms a phosphono-cholinesterase adduct. Some OP-modified cholinesterases are prone to non-reactivation, aging or other post-inhibitory mechanisms. This is important in certain nerve gas exposures in that “aging” is a determinant endpoint of the cholinesterase inhibition mechanism.
OP insecticides resemble OP nerve agents closely, except that they usually have a P═S bond instead of a P═O bond. OP insecticides generally require oxidative desulfuration to the P═O compound to exhibit maximum toxicity, but thereafter the chemical interaction with AchE is the same. It is known that replacing the P═O moiety with P═S generally reduces the reactivity of the OP (although biological oxidation to the P═O compound restores reactivity). When potentially reactive ethoxy or methoxy groups are replaced with their corresponding alkyl analogs (i.e., propyl or ethyl, respectively), the potential toxicity of the resulting compounds also decreases. Thus, the phosphonylated serine residues of AchE and BuChE are highly information rich molecules and indicate the type and amount of OP exposure whether it is from a nerve agent or a pesticide. A method of detection that may identify the precise OP agent (or agents) and exposure so that appropriate treatment and response can be taken is needed.
Human serum albumin (hSA) makes up 50-60% of serum proteins. hSA possesses an esterase and amidase activity. It has also been shown that hSA has the ability to bind OP agents. Binding of OPs to hSA occurs at tyrosine 411. Such a property makes hSA a potential biomarker for detection of exposure to OP reagents.
Antibodies elicited against the OP adducts of ChE members and against hSA or other enzymes or binding proteins may be used to determine an exposure to OP. Individual OPs form adducts specific to that reagent. Therefore, antibodies against each OP-adducted protein provide important information in determining exposure to a particular OP. This knowledge can lead to faster treatment and fewer long-term adverse health effects.